Method Of Differentiation From Stem Cells To Hepatocytes

ABSTRACT

Disclosed are: a gene transduction method for use in the induction of the differentiation of stem cells such as ES cells or iPS cells into hepatocytes effectively; stem cells into each of which a gene useful for the induction of the differentiation into hepatocytes is introduced; and hepatocytes produced from stem cells each having the gene introduced therein. A specific gene can be introduced into stem cells such as ES cells or iPS cells using an adenovirus vector. The effective induction of the differentiation into hepatocytes can be achieved by introducing the gene. Specifically, the effective induction of the differentiation of stem cells such as ES cells or iPS cells into hepatocytes can be achieved by introducing at least one gene selected from HEX gene, HNF4A gene, HNF6 gene and SOX17 gene into the stem cells.

TECHNICAL FIELD

The present invention relates to a method of differentiation frompluripotent stem cells such as embryonic stem cells (hereinafter, alsoreferred to as “ES cells”) or induced pluripotent stem cells(hereinafter, also referred to as “iPS cells”) to hepatocytes. Thepresent invention also relates to stem cells containing an introducedgene that is useful for differentiation to hepatocytes.

This application claims the benefit of priority to Japan applications,JP-A Nos. 2009-247342, 2010-121282 and 2010-154225, which areincorporated herein by reference.

BACKGROUND ART

Pluripotent stem cells are undifferentiated cells that have pluripotencyand self-replication competence and are suggested to have potency torepair damaged tissues. For that reason, pluripotent stem cells havebeen studied intensively, as they are considered useful in the field ofscreening for therapeutic drugs for various diseases and also in thefield of regeneration medicine. Among the pluripotent stem cells, iPScells (induced pluripotent stem cells) are stem cells prepared bydedifferentiation of somatic cells, such as fibroblasts, by introductionof a particular transcription factor, such as OCT3/4, SOX2, KLF4 orC-MYC, into the cells. Theoretically, totipotent cells can inducedifferentiation into all tissues and organs including liver.

Formation of spheroids (embryoid bodies), addition of a humoral factorto the medium, and addition for example of a suitable extracellularmatrix, feeder cells and matrigel, as properly selected, were mainlystudied as the methods for induction of differentiation from pluripotentstem cells to hepatocytes. However, it was reported that these methodsdemand longer culture period and are extremely lower indifferentiation-inducing efficiency and that the hepatocytes thusobtained are also lower in drug-metabolizing enzyme activity (NonpatentLiteratures 1 to 4). For improvement of the differentiation-inducingefficiency, introduction of a gene playing an important role indifferentiation may be introduced, for example, into pluripotent stemcells but, because there is no established method of introducing such agene efficiently, there is almost no report on induction ofdifferentiation into hepatocytes by gene introduction. Fordifferentiation from stem cells to mature hepatocytes, the stem cellsshould differentiate via endodermal cells and hepatic stem cells intojuvenile hepatocytes. In each differentiation step, a humoral factor,such as activin A, basic fibroblast growth factor (bFGF), BMP4 (bonemorphogenetic protein 4), FGF4, retinoic acid or DMSO, is used in theculture system. In addition, transcription factors such as HEX, HNF4A,HNF6 and FOXA2 are reported to be required embryologically as thefactors for inducing differentiation to hepatocytes (NonpatentLiterature 5). For example, HEX gene is found to be generated, forexample, in thyroid gland, lung, liver, blood cells and vascularendothelial cells. HEX gene-deficient mice are found to have nohepatocyte and die approximately on the 10.5th day of viviparity(E10.5). The HEX gene is considered to regulate expression, for example,of GATA4, HNF4A and FGF receptor genes.

As described above, when FOXA2, a possible hepatocyte-specifictranscription factor, was introduced into ES cells in development, therewas observed differentiation from the ES cells to hepatocytes, but alsoto cells other than hepatocytes, as reported in literature (NonpatentLiterature 6). This fact indicates that FOXA2 is not a transcriptionfactor that can induce differentiation of pluripotent stem cells such asES cells only to hepatocytes, and the action of the factor is yet to beelucidated sufficiently.

A system employing an adenovirus (hereinafter, referred to simply as“Ad”) was developed as a vector system for next-generation gene therapy.Ad has a regular icosahedral structure consisting of 252 capsomeres, 12capsomeres thereof present on the vertices are called pentons, which hasa protruded structure (consisting of penton base and fiber), and theother 240 capsomeres are called hexons. A virus enters cell, as thefiber binds to the Ad receptor (CAR; coxsackievirus-adenovirus receptor)and then the RGD motif of penton base binds to intgrins (αvβ3 and αvβ5)on the cell surface. However, in later studies various studies aimed atmaking the Ad vector incorporated into cells expressing no or little CARwere made and reported (Patent Documents 1 to 3). There is a report ongene delivery to mesenchymal stem cells by using such an improved Advector (Nonpatent Literature 7). It is disclosed here that genes areintroduced for example into mesenchymal stem cells by using variousimproved Ad vectors. However, mesenchymal stem cells and ES cells arecompletely different from each other. In addition, the NonpatentLiterature only discloses that the Ad vector has a role of delivering agene into cells (Drug Delivery System). It was reported that Ad vectorscan be used as a tool for differentiation of mouse ES and iPS cells(Nonpatent Literatures 8 and 9). However, these Nonpatent Literatures donot disclose a method of inducing differentiation from stem cells tohepatocytes.

PRIOR ART LITERATURE Patent Documents

-   Patent Document 1: JP-ANo. 2002-272480 (Patent 3635462)-   Patent Document 2: JP-A No. 2003-250566-   Patent Document 3: JP-ANo. 2008-136381

Nonpatent Literatures

-   Nonpatent Literatures 1: Genes to Cells, 13, 731-746 (2008)-   Nonpatent Literatures 2: Stem Cells, 26, 894-902 (2008)-   Nonpatent Literatures 3: Stem Cells, 26, 1117-1127 (2008)-   Nonpatent Literatures 4: Hepatology, 45, 1229-1239 (2007)-   Nonpatent Literatures 5: Nature Reviews Genetics, 3, 499-215 (2002)-   Nonpatent Literatures 6: FASEB Journal, 16, 1444-1446 (2002)-   Nonpatent Literatures 7: Biochem. Biophys. Res. Commun., 332,    1101-1106 (2005)-   Nonpatent Literatures 8: Mol Ther., 12 (3), 547-554 (2005)-   Nonpatent Literatures 9: Stem Cells, 27 (8), 1802-1811 (2009)

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

An object of the present invention is to provide a method ofdifferentiation from stem cells to hepatocytes by introducing a geneinvolved in differentiation into pluripotent stem cells such as ES oriPS cells. Another object of the present invention is to provide a stemcell containing a gene useful for induction of differentiation intotissue cells and a hepatocyte generated by differentiation from the stemcells containing the gene introduced.

Means to Solve the Problems

After intensive studies to achieve the objects above, the inventors havefound that it is possible to introduce a particular gene intopluripotent stem cells such as ES or iPS cells by using an Ad vector andto induce differentiation to hepatocytes effectively by introducing aparticular gene using the vector, and made the present invention.

Specifically, the present invention includes the following aspects:

1. a method of differentiation from stem cells to hepatocytes,characterized by introducing a gene into stem cells using an Ad vector;2. the method of differentiation described in aspect 1, wherein the geneintroduced is one or more genes selected from HEX, HNF4A, HNF6 and SOX17genes;3. the method of differentiation described in aspect 1 or 2, wherein atleast the HEX gene is introduced and additionally the HNF4A gene and/orthe SOX17 gene are introduced;4. the method of differentiation described in aspect 3, wherein theSOX17 gene and then the HEX gene are introduced into stem cells.5. the method of differentiation described in aspect 3 or 4, wherein theHEX gene and then the HNF4A gene are introduced into stem cells;6. a method of differentiation from stem cells to hepatocytes,comprising the steps of:1) introducing an HEX gene cassette into an Ad vector;2) introducing the HEX gene into cells by bringing the Ad vectorcontaining the gene introduced in step 1) with stem cells; and3) culturing the stem cells containing the gene introduced therein;7. the method of differentiation from stem cells to hepatocytesdescribed in aspect 6, comprising, before the steps 1) to 3) above, thefollowing steps of:a) introducing an SOX17 gene cassette into an Ad vector;b) introducing the SOX17 gene into cells by bringing the Ad vectorcontaining the gene introduced in step a) with stem cells; andc) culturing the stem cells containing the gene introduced therein;8. the method of differentiation from stem cells to hepatocytesdescribed in aspect 6 or 7, comprising, after the step of culturing thestem cells containing the HEX gene introduced in step 3), a step ofintroducing an HNF4A gene into the cells additionally by bringing thecultured cell into contact with an Ad vector containing the HNF4A geneintroduced therein;9. the method of differentiation described in any one of aspects 1 or 8,wherein the stem cells are treated with a humoraldifferentiation-inducing factor;10. the method of differentiation described in any one of aspects 1 or9, wherein the stem cell is an embryonic stem cell or an inducedpluripotent stem cell;11, the differentiation-inducing method described in aspect 10, whereinthe embryonic stem cell or the induced pluripotent stem cell is a humanembryonic stem cell or a human induced pluripotent stem cell;12. a stem cell containing a gene introduced by the method ofdifferentiation described in to any one of aspects 1 to 11;13. a hepatocyte differentiated from the stem cell containing anintroduced gene described in aspect 12; and14. a method of using the hepatocyte described in aspect 13 forevaluation of pharmaceutical toxicity or pharmacokinetics.

Advantageous Effect of the Invention

It was found that it is possible, by using the Ad vector according tothe present invention, to introduce a gene involved in induction ofdifferentiation of pluripotent stem cells such as ES or iPS cellseffectively and to induce differentiation to hepatocytes. Specifically,it is possible to induce differentiation from stem cells such as ES oriPS cells to hepatocytes effectively by introducing one or more genesselected from HEX, HNF4A, HNF6 and SOX17 genes into the stem cells. Inparticular, it was confirmed that it is possible to inducedifferentiation from stem cells to hepatocytes effectively byintroducing HEX gene into stem cells. It was further confirmed that itis possible to induce differentiation from stem cells to hepatocytesmore effectively by introducing additionally other genes such as SOX17gene and/or HNF4A gene, as needed, according to the progress of celldifferentiation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a chart showing the scheme of the test on induction ofdifferentiation from iPS cells to hepatocytes by introducing an HEX geneinto human iPS cells with an Ad vector. (Examples 1 and 3)

FIG. 2 is photographs by immunostaining, showing progress ofdifferentiation from human iPS cells to hepatocytes. (Test Example 1-1)

FIG. 3 is a graph showing the results obtained when the progress ofdifferentiation to hepatocytes when human iPS cells were cultured undervarious culture conditions was monitored, as compared to expression ofα-fetoprotein (AFP) as indicator. (Test Example 1-2)

FIG. 4 is a graph showing the results obtained when the progress ofdifferentiation to hepatocytes when human iPS cells were cultured undervarious culture conditions was monitored, as compared to expression ofalbumin as indicator. (Test Example 1-2)

FIG. 5 is a graph showing the results obtained when the progress ofdifferentiation to hepatocytes when human ES cells were cultured undervarious culture conditions was monitored, as compared to expression ofAFP as indicator. (Test Example 2)

FIG. 6 is a graph showing the results obtained when the progress ofdifferentiation to hepatocytes when human ES cells were cultured undervarious culture conditions was monitored, as compared to expression ofalbumin as indicator. (Test Example 2)

FIG. 7 is photographs by immunostaining, showing progress ofdifferentiation from human iPS cells to hepatocytes. (Test Example 3-1)

FIG. 8 is a chart showing the scheme of the test on induction ofdifferentiation from iPS cells to hepatocytes by introducing an HEX geneinto human iPS cells with an Ad vector. (Example 4)

FIG. 9 is a graph showing the results obtained when the progress ofdifferentiation to hepatocytes when human iPS cells were cultured undervarious culture conditions was monitored, as compared to expression ofAFP and albumin as indicators. (Test Example 4-1)

FIG. 10 is photographs by immunostaining, showing progress ofdifferentiation from human iPS cells to hepatocytes. (Test Example 4-2)

FIG. 11 is a chart showing the scheme of the test on induction ofdifferentiation from iPS cells to hepatocytes by introducing an HEX geneinto human iPS cells with an Ad vector. (Example 5)

FIG. 12 is graphs showing the results obtained when the expressionamounts of endoderm markers FOXA2 and SOX17 when 3 kinds of human stemcells were cultured were monitored. (Test Example 5-1)

FIG. 13 is graphs showing the results obtained when progress ofdifferentiation to hepatocytes when 3 kinds of human stem cells werecultured was monitored, as compared to expression of α-fetoprotein (AFP)and albumin as indicators. (Test Example 5-1)

FIG. 14 is a chart showing the scheme of the test on induction ofdifferentiation from iPS cells to hepatocytes by introducing an HEX geneinto human iPS cells with an Ad vector. (Example 6)

FIG. 15 is photographs by immunostaining, showing progress ofdifferentiation from human iPS cells to hepatocytes. (Test Example 6-1)

FIG. 16 is a chart showing the scheme of the test on induction ofdifferentiation from iPS cells to hepatocytes by introducing an HEX geneinto human iPS cells with an Ad vector. (Examples 7 and 8)

FIG. 17 is a graph showing the results obtained when the function of theHEX gene-introduced human iPS cells (Tic cell line) was analyzed byexpression of drug-metabolizing enzyme cytochrome P450 3A4 (GYP3A4).(Test Example 7-1)

FIG. 18 is a graph showing the results obtained when the function of theHEX gene-introduced human iPS cells was analyzed by function ofdrug-metabolizing enzyme cytochrome P450 3A4 (CYP3A4) and drug response.(Test Example 7-2)

FIG. 19 is graphs showing the results when progress of differentiationfrom HEX gene-introduced human iPS cells (Tic cell line and Dotcom cellline) to hepatocytes was analyzed as compared to expression of albuminas indicator and function of the cells analyzed by expression ofdrug-metabolizing enzyme cytochrome P450 3A4 (CYP3A4). (Test Example8-1)

FIG. 20 is a chart showing the scheme of the test on induction ofdifferentiation from iPS cells to hepatocytes by introducing an HEX geneinto human iPS cells with an Ad vector. (Example 9)

FIG. 21 is photographs showing the morphological change of the HEXgene-introduced human iPS cells (Tic cell line). (Test Example 9-1)

FIG. 22 is a chart showing the scheme of the test on induction ofdifferentiation from iPS cells to hepatocytes by introducing SOX17 andHEX genes into human iPS cells with an Ad vector. (Example 10)

FIG. 23 is photographs by immunostaining, showing progress ofdifferentiation from human iPS cells to hepatocytes. (Test Example 10-1)

FIG. 24 is graphs showing the results obtained when progress ofdifferentiation to hepatocytes, when human iPS cells were cultured undervarious conditions, was monitored, as compared with expression of AFPand albumin as indicators. (Test Example 10-2)

FIG. 25 is a chart showing the scheme of the test on induction ofdifferentiation from iPS cells to hepatocytes by introducing HEX andHNF4A genes into human iPS cells with an Ad vector. (Example 11)

FIG. 26 is photographs by immunostaining, showing expression ofdrug-metabolizing enzyme cytochrome P450 3A4 (CYP3A4) in human iPS cellscontaining each gene introduced thereto. (Test Example 11-1)

FIG. 27 is graphs showing the results when the function of the HEX andHNF4A gene-introduced human iPS cells was analyzed, as compared toexpression of drug-metabolizing enzyme cytochromes P450 2D6 (CYP2D6),3A4 (CYP3A4), and 7A1 (CYP7A1). (Test Example 11-2)

FIG. 28 is a graph showing the results when the function of the HEX andHNF4A gene-introduced human iPS cells was analyzed by drug-metabolizingactivity. (Test Example 11-3)

FIG. 29 is a chart showing the scheme of the test on induction ofdifferentiation from iPS cells to hepatocytes scheme by introducingSOX17, HEX and HNF4A genes into human iPS cells with an Ad vector.(Example 12)

FIG. 30 is graphs showing the results when the function of the human iPScells containing one or more genes selected from SOX17, HEX and HNF4Agenes was analyzed, as compared to expression of drug-metabolizingenzyme cytochromes P450 2D6 (CYP2D6), 3A4 (CYP3A4) and 7A1 (CYP7A1).(Test Example 12)

FAVORABLE EMBODIMENTS OF THE INVENTION

The present invention relates to a method of introducing a gene intostem cells by using an Ad vector and thus inducing differentiation fromstem cells to hepatocytes. In the present invention, the stem cell is apluripotent stem cell such as ES cell or iPS cell, particularlyfavorably a human ES cell or a human iPS cell, but cells that candifferentiate into the directed hepatocyte by introduction of a geneinto human ES or iPS cells, such as mesendodermal, endodermal andhepatic stem cells, are also included. Examples of the hepatocytesaccording to the present invention, more specifically hepatocytesdifferentiated from the stem cells containing an introduced gene,include mature hepatocytes and also cells can differentiate intodirected hepatocytes by introduction of a gene into stem cells, such ashepatic stem cells and juvenile hepatocytes.

The ES cell is a pluripotent stem cell obtained by in vitro culture of acell mass, called inner cell mass, that is present in the embryo at theblastocyst stage and isolation from the culture as undifferentiated stemcell population. The ES cell was established as a cell line pluripotentin mouse by M. J. Evans & M. H. Kaufman (Nature, 292, 154, 1981) andthen by G. R. Martin (Natl. Acad. Sci. USA, 78, 7634, 1981). There aremany human-derived ES cell lines already established and these celllines can be obtained, for example, from ES Cell International,Wisconsin Alumni Research Foundation, National Stem Cell Bank (NSCB) andothers. An ES cell is generally established by culture of an earlyembryonic cell, but it is possible to prepare an ES cell from an earlyembryo containing the nucleus of somatic cell transplanted.Alternatively, a cell structure like the embryo at the blastocyst stagemay be produced by transplanting the cell nucleus of a desired animalinto cytoplasts or ooplastoids prepared by dividing egg cells ordenucleated egg cells of a foreign animal into multiple pieces and an EScell may be produced based on the cell. In addition, studies onpreparation of ES cells by developing a parthenogenetic embryo to thephase similar to blastocyst stage and preparing an ES cell from theembryo and also on preparation of ES cells having genetic information ofsomatic cell nucleus by fusion of ES and somatic cells are alsoreported. The ES cell used in the present invention may be an ES cellprepared by such a known method or an ES cell that is prepared by a newmethod that will be developed in the future.

Alternatively, the iPS cell is an induced pluripotent stem cell that ispluripotent and proliferating similarly to ES cells that was preparedwithout use of eggs, embryos or ES cells by reprogramming differentiatedcell by introduction of several kinds of genes into a somatic cell andwas established from a mouse fibroblast in 2006 for the first time inthe world. It was also reported that human iPS cells were successfullyestablished with four human genes OCT3/4, SOX2, KLF4 and C-MYChomologous to the four genes used in establishing mouse iPS cells (Cell131: 861-872, 2007) introduced in a human fibroblast. The iPS cell foruse in the present invention may be an iPS cell prepared by such a knownmethod or an iPS cell that is prepared by a new method developed in thefuture.

The method of culturing the stem cells such as ES or iPS cells is notparticularly limited and may be any known method. A known medium or anew medium to be developed in the future may be used as the medium forkeeping the ES cells in the undifferentiated and pluripotent state andalso as the medium suitable for induction of differentiation. Typicalexamples thereof include commercially available basal media for mammalcells, such as DMEM and/or DMEM/F12, containing serum or knock-out serumreplacement (KSR) and bFGF and others added thereto, commerciallyavailable media for primate ES cells, a basal medium hESF-GRO forproliferation of primate ES cells, a basal medium hESF-DIF for inductionof differentiation of primate ES cells, and a medium CSTI-7 forproliferation of primate ES cells. The medium may contain knownadditives suitable for use in culture of pluripotent stem cells such asES or iPS cells, for example one or more additives selected from N2supplement, B27 supplement, insulin, bFGF, activin A, heparin, variousinhibitors such as ROCK inhibitor and GSK-3 inhibitor and others, atsuitable concentrations. The media and the additives can be used, asthey are selected properly for example according to the cells used andthe differentiation stage. For example, they may be selected by themethod described in Tiss, Cult. Res. Commun., 27: 139-147 (2008).

The Ad vector used in the present invention is not particularly limitedand an Ad vector prepared by a known method may be used. For example, itmay be an improved Ad vector improved for introduction thereof intocells expressing no or very limited CAR or an Ad vector for introductionthereof into cells expressing CAR. Specifically, it is possible to useAd vectors containing a DNA coding an cell adhesion peptide (RGDsequence), a representative of adhesion peptides, a DNA coding a peptidecompatible with heparan sulfate (K7 (KKKKKKK) sequence), a DNA coding apeptide compatible with laminin receptor, or a DNA coding a peptidecompatible with E-selectin, and also the Ad vectors for example shown inPatent Documents 1 to 3.

A desired gene shown below can be introduced into the Ad vector by anyknown method or any method to be developed in the future. Such a genecan be introduced into the Ad vector according to the present inventionby cleaving the vector with one or more restriction enzymes at therespective recognition sequences, introducing the gene there with orwithout a shuttle vector and ligating the product in vitro.

In the present invention, the gene to be introduced by using the Advector may be any gene that can induce differentiation of pluripotentstem cells such as ES or iPS cells to hepatocytes effectively.Specifically, it is possible to introduce a gene selected from HEX,HNF4A, HNF6 and SOX17 genes into stem cells, and in particular,introduction of HEX gene is favorable (see FIGS. 1, 8, 11, 14, 16 and20). Introduction of the HEX gene results in induction ofdifferentiation from stem cells to hepatocytes. For effective inductionof differentiation to hepatocytes, it is favorable to introduce theSOX17 gene first, determining the directing of differentiation from stemcells to endodermal cells, and introduce the HEX gene then (see FIG.22). It is also possible to determine the directing of differentiationto hepatocytes by introducing the HEX gene and then promotedifferentiation to hepatocytes by introducing a gene selected from HNF4Agene, HNF6 gene and FOXA2 gene, particularly preferably HNF4A gene (seeFIG. 25). For the most effective induction of differentiation from stemcells to hepatocytes, it is considered most preferable to introduce theSOX17 gene, thus determining the directing of differentiation from stemcells to hepatocytes, and the HEX gene and additionally the HNF4A gene(see FIG. 29). For example, it is possible to induce differentiationfrom ES or iPS cells to hepatocytes by the operations in the followingSteps 1) to 3):

1) a step of introducing a gene that can effectively inducedifferentiation from stem cells such as ES or iPS cells to hepatocytesinto an Ad vector,

2) a step of bringing the Ad vector having the gene introduced thereinprepared in step 1) above in contact with stem cells and thusintroducing a gene that can effectively induce differentiation from stemcells to hepatocytes into the stem cells, and

3) a step of culturing the gene-containing stem cells.

In the step 1), the gene that can effectively induce differentiationfrom stem cells to hepatocytes is a gene selected from HEX, HNF4A andHNF6 genes, more preferably HEX gene. It is possible to directdifferentiation to hepatocytes more effectively by introducing the SOX17gene into the stem cells before introduction of the gene. Further, it ispossible to induce differentiation to hepatocytes more effectively, byintroducing a gene selected from HNF4A, HNF6 and FOXA2 genes,particularly favorably HNF4A gene into the cultured cells after step 3).It would be possible to prepare hepatocytes from iPS cells efficiently,by introducing, in addition to SOX17, HEX and HNF4A, one or more genesthat are involved in differentiation and proliferation of hepatocytes,such as GATA4, GATA6, HNF1A, HNF1B, FOXA1/HNF3A, FOXA2/HNF3B,FOXA3/HNF3G CEBPA, CEBPB, TBX3 and PROX1. The step of introducing eachgene can be selected properly according to the differentiation stage ofthe cells. For preparation of biliary epithelial cells surroundinghepatocytes, it would be effective to introduce Sa114 and HNF6 genes.These genes are introduced into the stem cells, by the Ad vectorscontaining desired genes into contact with the stem cells according tothe differentiation stage of the cells.

As for the genes to be introduced, the HEX gene used may be, forexample, the gene of GenBank Accession No. BC014336; the HNF4A geneused, the gene of GenBank Accession No. NM000457; the HNF6 gene used,the gene of GenBank Accession No. NM004498; the FOXA2 gene used, thegene of GenBank Accession No. BC011780; and the SOX17 gene used, thegene of GenBank Accession No. NM_022454.

The Ad vector according to the present invention can be prepared by amethod comprising the following steps A) and B):

A) a step of constructing an expression construct containing a promotersequence in the noncoding region of the introduced gene, andB) a step of cleaving the Ad genome with a restriction enzyme andligating the expression construct prepared in step A) into the Adgenome.

The Ad vector according to the present invention may be prepared byconstructing a shuttle vector containing the expression construct ofstep A) and ligating the gene expression shuttle vector into the Adgenome between the steps A) and B) above.

It is known in the art that humoral factors such as activin A, bFGF,BMP4 and FGF-4 are used in the differentiation steps fromundifferentiated cells to mature hepatocytes. The humoral factors may bebrought into contact with the cells in combination also in the step ofinduction of differentiation from stem cells such as ES or iPS cells tohepatocytes according to the present invention. The step of bringing thehumoral factors into contact with the cells is not particularly limitedand may be carried out before or after introduction of the variousselected genes above. If multiple genes are to be introduced, thehumoral factors may be brought into contact with the cells between theintroduction steps of respective genes. Adhesion between cells orbetween cells and other cells or substrate is needed for growth anddivision of normal cells, and proteins (extracellular matrix) presentbetween cells or between the cells and the substrate are also needed. Inthe method of inducing differentiation to hepatocytes according to thepresent invention, the extracellular matrices described above may beadded in the steps above. Examples of the extracellular matrices includematrigel, fibronectin, vitronectin, laminin, nidogen, tenascin,thrombospondin, type-I collagen, type-IV collagen, gelatin, andsynthetic substrates equivalent thereto, and in the present invention,matrigel or laminin can be used particularly favorably. Specifically,they can be used, as they are coated on or added to a container forculture.

The present invention also relates to a stem cell such as ES or iPS cellcontaining an Ad vector having the gene introduced therein. It alsorelates to a hepatocytes generated from the stem cell containing the Advector having the gene introduced therein.

It is possible by using hepatocytes prepared by the method of inducingdifferentiation according to the present invention to examinepharmaceutical toxicity and pharmacokinetics. Specifically, it ispossible to predict the toxicity of a candidate drug compound in thebody by adding the candidate drug compound to the hepatocytes preparedaccording to the present invention and analyzing expression andfluctuation of hepatotoxicity markers. It is also possible to predictthe pharmacokinetics (metabolite) of a candidate drug compound in thebody by adding the candidate drug compound to the hepatocytes preparedaccording to the present invention and analyzing the metabolites of thecompound. It is possible in this way to screen candidate drug compoundsto be eliminated because of problems in toxicity and pharmacokineticsrapidly and thus to accelerate drug development. The present inventionalso relates to a method of using the hepatocytes prepared by the methodof inducing differentiation according to the present invention forevaluation of pharmaceutical toxicity and pharmacokinetics.

EXAMPLES

Hereinafter, the present invention will be described more specificallywith reference to Examples and test Examples for deeper understanding ofthe present invention, but it should be understood that the scope of thepresent invention is not restricted by these examples.

Various media are needed for human iPS or ES cells in Examples and TestExamples.

Compositions of the various media used will be described in ReferenceExample below and the differentiation-inducing methods then in theExamples below.

Reference Example Composition of Various Media

1) A medium in the composition shown in Table 2 of Tiss. Cult. Res.Commun., 27: 139-147 (2008), wherein the basic medium is knock-outDMEM/F12 and which contains bFGF (10 ng/ml), was used as the medium forkeeping human iPS cells undifferentiated. Hereinafter, the medium willbe referred to as “medium 1.”

2) A medium in the composition shown in Table 2 of Tiss. Cult. Res.Commun., 27: 139-147 (2008), wherein basic medium is DMEM/F12 and whichcontains bFGF (5 ng/ml), was used as the medium for keeping human EScells undifferentiated. Hereinafter, the medium will be referred to as“medium 2.”

3) A basic medium for culturing human ES cells, i.e., hESF-GRO medium(Cell Science & Technology Institute, Inc.), containing insulin (10μg/ml), transferrin (5 μg/ml), albumin conjugate oleic acid (9.4 μg/ml),2-mercaptoethanol (10 μM), 2-ethanolamine (10 μM), sodium selenite (20nM), heparin (100 ng/ml) and bFGF (10 ng/ml) (Proc. Natl. Acad. Sci.USA; 105 (36): 13409-13414 (2008)) was used as the medium for keepingcells undifferentiated. Hereinafter, the medium will be referred to as“medium 3.” The medium 3, which demands no feeder cell or Knock-outSerum Replacement (KSR), permits culture of human iPS cells, as thecells are kept pluripotent in the undifferentiated state.

4) A basic medium for culturing human ES cells, i.e., hESF-GRO (CellScience & Technology Institute, Inc.) containing insulin (10 μg/ml),transferrin (5 μg/ml), 2-mercaptoethanol (10 μM), 2-ethanolamine (10μM), sodium selenite (20 nM) and bovine serum albumin (BSA) (1 mg/ml)was used as the medium for inducing differentiation. Hereinafter, themedium will be referred to as “medium 4.”

5) A basic medium for inducing differentiation of human ES cells, i.e.,hESF-DIF (Cell Science & Technology Institute, Inc.) containing insulin(10 μg/ml), transferrin (5 μg/ml), 2-mercaptoethanol (10 μM),2-ethanolamine (10 μM), sodium selenite (20 nM) (FASEB J. 23: 114-22(2009)) was used as another embodiment of the medium for inducingdifferentiation. Hereinafter, the medium will be referred to as “medium5.”

Example 1 Method of Inducing Differentiation from Human iPS Cells toHepatocytes

In this Example, induction of differentiation from human iPS cellscontaining the HEX gene introduced by using Ad vector to hepatocyteswill be described. The experimental scheme in this Example is shown inFIG. 1.

1) Construction of Ad Vector for Introduction of HEX Gene

An Ad vector having a gene introduced into the E1-deficient region of aE1-deficient type-5 Ad genome (pAdHM41-K7) and an EF-1α promoter in theregion upstream thereof was prepared. The gene introduced was the HEXgene having the sequence of GenBank Accession No. BG014336.

2) Culture of Human iPS Cells

Human iPS cells (Tic (JCRB1331), Dotcom (JCRB1327), and Squeaky(JCRB1329)) were used in this Example. Cultured human iPS cells wereobtained by using mouse fetal fibroblasts (MEF) as feeder cells and themedium 1 shown in Reference Example according to the method described inTiss. Cult. Res. Commun., 27:139-147 (2008). The culture medium wasreplaced with the medium 3 shown in Reference Example, two days beforeinduction of differentiation of the human iPS cells.

The culture solution was then treated with a cell dissociation solutionAccutase® (Invitrogen) at 37° C. for 3 minutes and the supernatantcontaining floating feeder cells was removed. Human iPS cells were thencollected by using the medium 3 and the cell suspension was centrifugedat 1,300 rpm for 3 minutes. The cells were resuspended in the medium 4shown in Reference Example and centrifuged at 1,300 rpm for 3 minutesadditionally twice.

The human iPS cells obtained by centrifugation were resuspended in themedium 4 containing 50 ng/ml of activin A (R&D Systems) and 10 ng/ml ofbFGF (R&D Systems); the human iPS cells were inoculated in each well ofa laminin (sigma)-coated cell culture plate (12 wells) in an amount of2.5×10⁵ cell/well; the medium was replaced every day with the medium 4containing 50 ng/ml of activin A (R&D Systems) and 10 ng/ml of bFGF (R&DSystems) cultured at 37° C. The day when the human iPS cells wereinoculated is the 0th day of differentiation induction.

3) Introduction of HEX Gene into Human iPS Cells

On the 5th day from differentiation induction, the human iPS cells wereseparated from the cell culture plate with 0.0125% trypsin-0.01325 mMEDTA; and after trypsin was neutralized with 0.1 mg/ml trypsininhibitor, the cells were centrifuged from the medium 5 shown inReference Example twice at 1,300 rpm for 3 minutes. The human iPS cellsobtained after centrifugation was resuspended in the medium 5 containing50 ng/ml of activin A (R&D Systems) and 10 ng/ml of bFGF (R&D Systems),inoculated in each well of a laminin (sigma)-coated cell culture plate(12 wells) in an amount of 5.0×10⁵ cell/well and cultured at 37° C.

After 24 hours, the cells were infected with the AdK7 (Ad vectorcontaining K7 peptide in the fiber knob region) vector containing theHEX gene introduced in step 1) at a concentration of 3,000 VP (vectorparticle)/cell for 1.5 hours, and the medium was replaced with themedium 5 containing 10 ng/ml of BMP4 (R&D Systems) and 10 ng/ml of FGF-4(R&D Systems) added thereto. The medium was then replaced every day withthe medium 5 containing 10 ng/ml of MBP4 (R&D Systems) and 10 ng/ml ofFGF-4 (R&D Systems) added thereto.

Test Example 1-1 Results Obtained by Immunostaining Method

On the 12th day of culture, expression of each gene was examined by animmunostaining method using Alexa®594-labeled anti-α-fetoprotein (AFP)antibody (manufactured by Dako) and Alexa®488-labeled anti-CK7 antibody(manufactured by Invitrogen). AFP is a marker for hepatic stem cells andCK7, a marker for biliary epithelial cells. As shown in FIG. 2, it wasfound that both markers reacted significantly in the systems containingthe HEX gene introduced with the Ad vector than in the systems withoutthe gene introduced. The results show that introduction of HEX geneusing the Ad vector leads to enhanced induction of differentiation fromhuman iPS cells to hepatocytes.

Test Example 1-2 Results Obtained by Real-Time PCR Method

The expression amounts of possible hepatic stem cell markers AFP andalbumin were determined on the 0th day of culture (undifferentiatedcell), 5th day (endodermal cell) and 12th day by a real-time PCR methodof using TaqMan® Gene Expression Assays (Applied Biosystems, catalognumber: Hs01040607_1 for AFP and Hs00910225_m for albumin). Theexpression amounts were calculated, based on the standard (100) of theexpression amount of each gene in Human Fetal Liver Total RNA (Clontech,catalog number: 636540).

The results showed definitely that both AFP and albumin were expressedin greater amounts in the cells overexpressing the HEX gene and thus,differentiation was induced more intensively in the HEXgene-overexpressing cells (FIGS. 3 and 4).

Example 2 Method of Differentiation from Human ES Cells to Hepatocytes

In this Example, HEX gene was introduced by using an Ad vector into thehuman ES cells (KhES-1) established by the Inst. for Frontier MedicalScience, Kyoto Univ., replacing the human iPS cells above, and inductionof differentiation from human ES cells to hepatocytes was studied. Inthis Example, 1) an Ad vector for introduction of HEX gene wasconstructed, 3) the HEX gene was introduced into human ES cells, and thetest was performed in a manner similar to Example 1, except that thecultured human ES cells were prepared by using mouse fetal fibroblasts(MEF) as the feeder cells and the medium 2 shown in Reference Exampleaccording to the method described in Tiss. Cult, Res. Commun., 27:139-147 (2008).

Test Example 2 Results Obtained by Real-Time PCR Method

The expression amounts of AFP and albumin, indicators ofdifferentiation, were determined by a real-time PCR method on the 0thday of culture (undifferentiated cell), 5th day (endodermal cell) and12th day by the method similar to that in Example 1-2

The results showed definitely that both AFP and albumin were expressedin greater amounts in the system containing the HEX gene and thus,differentiation was induced significantly in HEX gene-containing systems(FIGS. 5 and 6).

Example 3 Method of Differentiation from Human iPS Cells to Hepatocytes

In this Example, induction of differentiation from human iPS cellscontaining the HEX gene introduced by using an Ad vector to hepatocyteswill be described. The experimental scheme in this Example is shown inFIG. 1. 1) Construction of an Ad vector for introduction of HEX gene, 2)culture of human iPS cells and 3) introduction of HEX gene into humaniPS cells were carried out in a manner similar to Example 1. Human iPScells (Squeaky (JCRB1329)) were used.

Test Example 3-1 Results Obtained by Immunostaining Method

On the 12th day of culture, expression of each gene of AFP and CK7 wasexamined by an immunostaining method using Alexa®594-labeled anti-AFPantibody (manufactured by Dako) and Alexa®488-labeled anti-CK7 antibody(manufactured by Invitrogen). AFP is a marker for hepatic stem cells andCK7 a marker for biliary epithelial cells. As shown in FIG. 7, it wasfound that both markers reacted significantly in the systems containingthe HEX gene introduced with the Ad vector than in the systems withoutthe gene introduced. The results show that introduction of HEX geneusing the Ad vector leads to enhanced induction of differentiation fromhuman iPS cells to hepatocytes.

Example 4 Method of Differentiation from Human iPS Cells to Hepatocytes

In this Example, induction of differentiation from human iPS cellscontaining the HEX gene introduced by using an Ad vector to hepatocyteswill be described. The experimental scheme in this Example is shown inFIG. 8. 1) Construction of an Ad vector for introduction of HEX gene, 2)culture of human iPS cells and 3) introduction of HEX gene into humaniPS cells were carried out in a manner similar to Example 1. Human iPScells (Tic (JCRB1331)) were used.

Test Example 4-1 Results Obtained by Real-Time PCR Method

The expression amounts of possible hepatic stem cell markers AFP andalbumin were determined on the 0th day of culture (undifferentiatedcell), 5th day (endodermal cell) and 12th day by a real-time PCR methodof using TaqMan® Gene Expression Assays (Applied Biosystems, catalognumber: Hs01040607_1 for AFP and Hs00910225_m for albumin). Theexpression amounts were calculated, based on the standard (100) of theexpression amount of each gene in Human Fetal Liver Total RNA (Clontech,catalog number: 636540).

The results showed definitely that both AFP and albumin were expressedin greater amounts in the cells overexpressing the HEX gene and thus,differentiation was induced more intensively in HEX gene-overexpressingcells (FIG. 9).

Test Example 4-2 Results Obtained by Immunostaining Method

On the 12th day of culture, expression of genes of AFP, albumin and CK7was determined by an immunostaining method, by using Alexa®594-labeledanti-AFP antibody (manufactured by Dako), Alexa®594-labeled anti-albumin(ALB) antibody (manufactured by SIGMA) and Alexa®488-labeled anti-CK7antibody (manufactured by Invitrogen). AFP is the marker for hepaticstem cells, albumin, the marker for hepatocytes, and CK7, the marker forbiliary epithelial cells. As shown in FIG. 10, it was found that thesemarkers reacted significantly in the cells overexpressing the HEX geneintroduced with the Ad vector than in the cells without the geneintroduced. The results show that introduction of HEX gene using the Advector leads to enhanced induction of differentiation from human iPScells to hepatocytes.

Example 5 Method of Induction of Differentiation from Human iPS and ESCells to Hepatocytes

1) Culture of Human iPS and ES Cells

In this Example, induction of differentiation from gene-containing humaniPS and ES cells, which are prepared by introducing the HEX gene intohuman iPS and ES cells using an Ad vector, to hepatocytes will bedescribed. The experimental scheme in this Example is shown in FIG.11. 1) Construction of an Ad vector for introduction of HEX gene, 2)culture of human iPS cells and 3) introduction of HEX gene into humaniPS cells were carried out in a manner similar to Example 1. The humaniPS cells used were Tic (JCRB1331) and Dotcom (JCRB1327) and the EScells used were khES1 ES cells.

Test Example 5-1 Results Obtained by Real-Time PCR Method

The expression amounts of the genes of possible endodermal cell markersFOXA2 and SOX17 and possible hepatic stem cell markers AFP and albuminin the cells on the 0th day of culture (undifferentiated cells), 6th day(endodermal cells) and 12th day were determined. Expression ofrespective genes was determined by a real-time PCR method of usingTaqMan® Gene Expression Assays (Applied Biosystems, catalog number:Hs00232764_m1 for FOXA2, Hs00751752_s1 for SOX17, Hs01040607 m1 for AFPand Hs00910225_m for albumin). The expression amounts were calculated,based on the standard (1) of the cells of human iPS cell Tic on the 0thday of culture (undifferentiated cells).

As shown in FIG. 12, the expression amounts of the endodermal cellmarkers FOXA2 and SOX17 were the largest in the cell line Dotcom on the6th day (endodermal cells). As shown in FIG. 13, the possible hepaticstem cell marker AFP and the possible hepatocyte marker albumin showedthe largest increase in expression amount in the cell line Dotcom whenthe HEX gene was introduced into the cells with an Ad vector. Theresults show that introduction of a HEX gene by using the Ad vectorleads to enhanced induction of differentiation from the cells expressingan endodermal marker significantly to hepatocytes.

Example 6 Effect of Induction of Hepatocyte Differentiation by HEX Gene(18th Day of Culture)

In this Example, induction of differentiation from human iPS cellscontaining a HEX gene introduced therein using an Ad vector tohepatocytes will be described. The experimental scheme in this Exampleis shown in FIG. 14. 1) Construction of an Ad vector for introduction ofHEX gene, 2) culture of human iPS cells and 3) introduction of HEX geneinto human iPS cells were carried out in a manner similar to Example 1.2) The human iPS cells were cultured by the following method:

2) Culture of Human iPS Cells

In this Example, human iPS cells (Tic (JCRB133D) were used. The cellswere cultured similarly to (Example 1) until the 9th day afterdifferentiation induction. The cells were then harvested with 0.0125%trypsin-0.01325 mM EDTA on the 9th day of culture and resuspended inHCM™ Hepatocyte Culture Medium (Lonza) containing SingleQuots® (Lonza),10 ng/ml of fibroblast growth factor 4 (FGF4), 10 ng/ml of hepatocytegrowth factor (HGF) (R&D Systems), 10 ng/ml of oncostatin M (R&DSystems) and 10⁻⁷ M dexamethasone (Sigma) added thereto, as described inStem Cells, 26: 1117-27 (2008), and the suspension was inoculated on atype-I collagen (Nitta Gelatin)-coated cell culture plate (12 wells).The medium was exchanged once a day.

Test Example 6-1 Results Obtained by Immunostaining Method

On the 18th day of culture, expression of respective genes wasdetermined by an immunostaining method by using Alexa®594-labeledanti-albumin (ALB) antibody (manufactured by SIGMA), Alexa®594-labeledanti-drug-metabolizing enzyme cytochrome P450 3A4 (CYP3A4) antibody(manufactured by Santa Cruz) and Alexa®594-labeledanti-drug-metabolizing enzyme cytochrome P450 7A1 (CYP7A1) antibody(manufactured by Santa Cruz). Albumin, CYP3A4 and VYP7A1 were allhepatocyte markers. As shown in FIG. 15, it was found that allhepatocyte markers reacted significantly in the systems containing theHEX gene introduced with the Ad vector than in the systems without thegene introduced on the 18th of culture.

Example 7 Evaluation of Functionality of HEX Gene-Introduced Cells

In this Example, induction of differentiation from human iPS cellscontaining a HEX gene introduced by using an Ad vector to hepatocyteswill be described. The experimental scheme in this Example is shown inFIG. 16. 1) Construction of an Ad vector for introduction of HEX geneand 3) introduction of HEX gene into human iPS cells were carried out ina manner similar to Example 1, while 2) culture of human iPS cells wascarried out in a manner similar to Example 6. Human iPS cells (Tic(JCRB1331)) were used in this Example.

Test Example 7-1 Results Obtained by Real-Time PCR Method

The expression amount of the drug-metabolizing enzyme cytochrome P4503A4 (CYP3A4) in the cells on the 18th day of culture was determined by areal-time PCR method by using TaqMan® Gene Expression Assays (AppliedBiosystems, catalog number: Hs00430021_m1 for CYP3A4). The expressionamount was calculated, based on the standard (100) of the expressionamounts of respective genes in Human Adult Liver Total RNA (Clontech,catalog number: 636531) (FIG. 17).

As a result, it was found that introduction of HEX gene with the Advector leads to drastic increase of the expression amount of CYP3A4 tothe amount equivalent to that of fetal hepatocytes.

Test Example 7-2 Measurement of Activity of Drug-Metabolizing EnzymeCytochrome P450 3A4 (CYP3A4)

25 μM rifampicin (Sigma) or DMSO was added to human iPS cell-derivedhepatocytes or human hepatoma cell line HepG2 on the 18th day afterdifferentiation induction and the activity of CYP3A4 was determined byusing P450-Glo™ CYP3A4 Assay Kit (Promega) after 72 hours. The activitywas determined quantitatively by using a luminometer (Lumat LB 9507,Berthold).

It was found that the activity of CYP3A4 increased and drug response torifampicin also increased in the system containing the HEX geneintroduced with the Ad vector, compared to the system without theintroduce gene on the 18th day of culture (FIG. 18)

Example 8 Induction of Differentiation from iPS Cell Line that EasilyDifferentiates into Endodermal Cells to Hepatocytes

In this Example, induction of differentiation from human iPS cellscontaining a HEX gene introduced by using an Ad vector to hepatocyteswill be described. The experimental scheme in this Example is shown inFIG. 16. 1) Construction of an Ad vector for introduction of HEX geneand 3) introduction of HEX gene into human iPS cells were carried out ina manner similar to Example 1, while 2) culture of human iPS cells wascarried out in a manner similar to Example 6. In this Example, human iPScells (Tic (JCRB1331) and Dotcom (JCRB1327)) were used.

Test Example 8-1 Measurement of Activity of Drug-Metabolizing EnzymeCytochrome P450 3A4 (CYP3A4)

The activity of CYP3A4 was determined in a manner similar to Example7-2. As a result, it was possible, by introducing the HEX gene with anAd vector, to induce differentiation from a cell line Dotcom higher inthe potential of differentiating into endodermal cells to hepatocytesthat express albumin and GYP3A4 in greater amounts (FIG. 19).

Example 9 Morphological Change During Differentiation Induction

In this Example, induction of differentiation from human iPS cellscontaining a HEX gene introduced by using an Ad vector to hepatocyteswill be described. The experimental scheme in this Example is shown inFIG. 20. 1) Construction of an Ad vector for introduction of HEX geneand 3) introduction of HEX gene into human iPS cells were carried out ina manner similar to Example 1, while 2) culture of human iPS cells wascarried out in a manner similar to Example 6. Human iPS cells (Tic(JCRB1331)) were used in this Example.

Test Example 9-1 Morphological Observation

The shape of the cells on the 0th day of culture (undifferentiatedcell), 6th day (endodermal cell), 9th day (hepatic stem cell) and 21stday was observed. As a result, the appearance of the cells changed fromthe shape of dense cells (0th day of culture) to the shapecharacteristic for the differentiated cells, in which the cellulardensity is low (6th day of culture). In addition, the cells in thesystem containing HEX gene introduced with an Ad vector changed intoepithelial cell-like shape characteristic to hepatocytes over the periodof 9th to 21st day of culture. Thus, the results of morphologicalobservation also suggested that the differentiated cells induced byintroduction of the HEX gene were hepatocytes (FIG. 21).

Example 10 Method of Differentiation from Human iPS Cells to Hepatocytesby Using SOX17 and HEX Genes in Combination

In this Example, SOX17 and HEX genes were introduced into human iPScells with an Ad vector and induction of differentiation from thegene-containing human iPS cells to hepatocytes will be described. Theexperimental scheme in this Example is shown in FIG. 22.

1) Construction of Ad Vector for Introduction of SOX17 Gene

For introduction of the SOX17 gene, an Ad vector having a geneintroduced in the E1-deficient region of E1-deficient type-5 Ad genome(pAdHM41-K7) and an EF-la promoter in the region upstream thereof wasprepared. The introduced gene was the SOX17 gene having the sequence ofGenBank Accession No. NM_022454. The Ad vector for introduction of HEXgene was prepared in a manner similar to Example 1.

2) Culture of Human iPS Cells.

Human iPS cells (201B7(JCRB)) were used in this Example. The cells werecultured similarly to the method of Example 1 until the 3rd day afterdifferentiation induction.

3) Introduction of SOX17 Gene into Human iPS Cells

After culture for 72 hours from induction of differentiation, the cellswere infected with the AdK7 vector containing SOX17 gene inserted instep 1) at 3,000 VP/cell for 1.5 hours, and the medium was replaced withthe medium 4 containing 50 ng/ml of activin A (R&D Systems) and 10 ng/mlof bFGF (R&D Systems) added thereto. The medium was then replaced withthe medium 4 containing 50 ng/ml of Activin A (R&D Systems) and 10 ng/mlof bFGF (R&D Systems) every day.

4) Introduction of HEX Gene into SOX17 Gene-Overexpressing Cells

On the 5th day after induction of differentiation, the human iPS cellswere separated with 0.0125% trypsin-0.01325 mM EDTA from the cellculture plate; trypsin was neutralized with 0.1 mg/ml trypsin inhibitor;and the suspension was centrifuged from the medium 5 shown in ReferenceExample at 1,300 rpm for 3 minutes. The human iPS cells obtained aftercentrifugation was resuspended into the medium 5 containing 50 ng/ml ofactivin A (R&D Systems) and 10 ng/ml of bFGF (R&D Systems), inoculatedin each well of a laminin (sigma)-coated cell culture plate (12 wells)at 5.0×10⁵ cell/well and cultured at 37° C. After culture for 24 hours,the cells were infected with the AdK7 vector containing the HEX geneinserted thereto at 3,000 VP/cell for 1.5 hours, and the medium wasreplaced with the medium 5 containing 10 ng/ml of BMP4 (R&D Systems) and10 ng/ml of FGF4 (R&D Systems). The medium was then exchanged with themedium 5 containing 10 ng/ml of BMP4 (R&D Systems) and 10 ng/ml of FGF4(R&D Systems) once a day.

Test Example 10-1 Results Obtained by Immunostaining Method

On the 9th and 12th days of culture, expression of respective genes wasdetermined by an immunostaining method by using Alexa®594-labeledanti-α-fetoprotein (AFP) antibody (manufactured by Dako),Alexa®594-labeled anti-albumin (ALB) antibody (manufactured by SIGMA)and Alexa®488-labeled anti-CK7 antibody (manufactured by Invitrogen).AFP is a marker for hepatic stem cells, albumin, a marker forhepatocytes, and CK7, a marker for biliary epithelial cells. As shown inFIG. 23, on the 9th day of culture, the hepatic stem cell marker reactedsignificantly more in the cells overexpressing SOX17 and HEX genes, asintroduced with the Ad vector, than in the cells overexpressing only theHEX gene introduced, but the biliary epithelial cell marker was notexpressed in neither of the cells. On the 12th day of culture, thehepatocyte marker was found to react significantly more in the cellsoverexpressing SOX17 gene and HEX gene, as introduced with the Advector, than in the cells overexpressing only the HEX gene introduced.

Test Example 10-2 Results Obtained by Real-Time PCR Method

The expression amounts of the possible hepatic stem cell markers AFP andalbumin in the cells on the 9th and 12th days of culture were determinedby a real-time PCR method by using TaqMan® Gene Expression Assays(Applied Biosystems, catalog number: Hs01040607_m1 for AFP andHs00910225_m for albumin). The expression amounts were calculated, basedon the standard (100) of the expression amount of each gene in HumanFetal Liver Total RNA (Clontech, catalog number: 636540). The resultsshow that introduction of SOX17 and HEX genes with the Ad vector leadsto increased induction of differentiation from human iPS cells tohepatocytes, compared to the case when only the HEX gene was used (FIG.24).

Example 11 Method of Differentiation from Human iPS Cells to Hepatocytesby HEX and HNF4A Genes in Combination

In this Example, induction of differentiation from human iPS cellscontaining HEX and HNF4A genes introduced by using an Ad vector tohepatocytes will be described. The experimental scheme in this Exampleis shown in FIG. 25.

1) Construction of Ad Vector for Introduction of HNF4A Gene

An Ad vector having the gene introduced in the E1-deficient region ofE1-deficient type-5 Ad genome (pAdHM41-K7) and an EF-1α promoter in theregion upstream thereof was prepared. The introduced gene was the HNF4Agene having the sequence of GenBank Accession No. NM_000457. The Advector for introduction of HEX gene was prepared in a manner similar toExample 1.

2) Culture of Human iPS Cells

Human iPS cells (Tic (JCRB1331), Dotcom (JCRB1327), and 201B7) were usedin this Example. The cells were cultured in a manner similar to Example1 until the 9th day from induction of differentiation.

3) Introduction of HEX and HNF4A Genes into Human iPS Cells

On the 9th day from induction of differentiation, the cells wereinfected with the AdK7 vector containing the HNF4A gene inserted instep 1) at 3,000 VP/cell for 1.5 hours, and the medium was replaced withthe HCM™ medium (Lonza) containing 10 ng/ml of hepatocyte growth factor(HGF) (R&D Systems), 10 ng/ml of oncostatin M (R&D Systems), and 10⁻⁷ Mdexamethasone (Sigma). The medium was then exchanged with the HCM™medium (Lonza) containing 10 ng/ml of HGF (R&D Systems), 10 ng/ml ofoncostatin M (R&D Systems) and 10⁻⁷ M dexamethasone (Sigma) once everytwo days.

Test Example 11-1 Results Obtained by Immunostaining Method

On the 18th day of culture, expression of respective genes weredetermined by an immunostaining method by using Alexa®594-labeledanti-drug-metabolizing enzyme P450 2D6 (CYP2D6) antibody (manufacturedby Santa Cruz), Alexa®594-labeled anti-drug-metabolizing enzyme P450 3A4(CYP3A4) antibody (manufactured by Santa Cruz), and Alexa®594-labeledanti-drug-metabolizing enzyme P450 7A1 (CYP7A1) antibody (manufacturedby Santa Cruz). CYP2D6, CYP3A4 and CYP7A1 are all hepatocyte markers. Asshown in FIG. 26, on 18th day of culture, all hepatocyte markers werefound to react significantly more in the cells overexpressing HEX geneand HNF4A gene, as introduced with the Ad vector, than in the cellsoverexpressing only the HEX gene introduced,

Test Example 11-2 Results Obtained by Real-Time PCR Method

The expression amounts of the possible hepatocyte markers CYP2D6, CYP3A4and CYP7A1 in the cells on the 18th day of culture were determined by areal-time PCR method by using TaqMan® Gene Expression Assays (AppliedBiosystems, catalog number: Hs02576168_g1 for CYP2D6, Hs00430021_m1 forGYP3A4 and Hs00167982_m1 for CYP7A1). The expression amounts werecalculated, based on the standard (100) of the expression amount of eachgene in Human Adult Liver Total RNA (Clontech, catalog number: 636531).The results show that introduction of HEX and NF4A genes with the Advector leads to drastically increase of the expression amount of CYP2D6,CYP3A4 and CYP7A1, compared to the case when only the HEX gene was used,and thus it was possible to obtain hepatocytes higher in the expressionamounts of medicine metabolizing enzymes than normal cells (FIG. 27).

Test Example 11-3 Measurement of Activity of Drug-Metabolizing EnzymeP450 3A4

25 μM rifampicin (Sigma) or DMSO was added to hepatocyte or humanhepatoma cell line HepG2 derived from the human iPS cells on the 18thday from induction of differentiation and the activity of CYP3A4 wasdetermined by using P₄50-Glo™ CYP3A4 Assay Kit (Promega) after 72 hours.The activity was determined quantitatively by using a luminometer (LumatLB 9507, Berthold). The results showed that introduction of HEX andHNF4A genes with the Ad vector leads to increase of the activity ofCYP3A4 and also increase in drug response to rifampicin, compared to thecase where only the HEX gene was used. It was thus possible to obtainhepatocytes higher in the activity of drug-metabolizing enzyme thannormal cells, by introduction of the HEX and HNF4A genes with the Advector by the method above (FIG. 28).

Example 12 Method of Differentiation from Human iPS Cells to Hepatocytesby Introducing SOX17, HEX and HNF4A Genes in Combination

In this Example, induction of differentiation to hepatocytes containingSOX17, HEX and HNF4A genes introduced as needed by using an Ad vectorwill be described. The experimental scheme in this Example is shown inFIG. 29.

1) Ad Vector for Introduction of SOX17, HEX and HNF4A Genes

An Ad vector similar to that in Example 1 was used.

2) Culture of Human iPS Cells

Human iPS cells (Tic (JCRB1331)) were used in this Example. The cellswere cultured in a manner similar to Example 1 until the 3rd day afterinduction of differentiation.

3) Introduction of SOX17 Gene

On the 3rd day from induction of differentiation, the cells wereinfected with the AdK7 vector containing the SOX17 gene inserted theretoat 3,000 VP/cell for 1.5 hours, and the medium was replaced with themedium 4 containing 50 ng/ml of activin A (R&O Systems) and 10 ng/ml ofbFGF (R&D Systems). The medium was then replaced with the medium 4containing 50 ng/ml of activin A (R&D Systems) and 10 ng/ml of bFGF (R&DSystems) once a day. The cells without SOX17 gene introduced were usesas a control group.

4) Introduction of HEX Gene

On the 6th day after induction of differentiation, the cells wereseparated from the cell culture plate with 0.0125% trypsin-0.01325 mMEDTA; trypsin was neutralized with 0.1 mg/ml trypsin inhibitor; and thecells were centrifuged at 1,300 rpm for 3 minutes twice by using themedium 5 shown in Reference Example. The human iPS cells obtained aftercentrifugation were resuspended in the medium 5 containing 50 ng/ml ofactivin A (R&D Systems) and 10 ng/ml of bFGF (R&D Systems) addedthereto, inoculated in each well on a laminin (sigma)-coated cellculture plate (12 wells) in an amount of 5.0×10⁵ cell/well and culturedat 37° C. After 24 hours, the cells were infected with the AdK7 vectorcontaining the inserted HEX gene at 3,000 VP/cell for 1.5 hours and themedium was replaced with the medium 5 containing 10 ng/ml of BMP4 (R&DSystems) and 10 ng/ml of FGF4 (R&D Systems). The medium was thenexchanged with the medium 5 containing 10 ng/ml of BMP4 (R&D Systems)and 10 ng/ml of FGF4 (R&D Systems) added thereto once a day. The cellswithout the HEX gene introduced were used as a control group.

5) Introduction of HNF4A Gene

On the 9th day after induction of differentiation, the cells wereinfected with the AdK7 vector containing the HNF4A gene inserted instep 1) above at 3,000 VP/cell for 1.5 hours and the medium was replacedwith the HCM™ medium (Lonza) containing 10 ng/ml of HGF (R&D Systems),10 ng/ml of oncostatin M (R&D Systems) and 10⁻⁷ M dexamethasone (Sigma)added thereto. The medium was then exchanged with the HCM™ medium(Lonza) containing 10 ng/ml of HGF (R&D Systems), 10 ng/ml of oncostatinM (R&D Systems) and 10⁻⁷ M dexamethasone (Sigma) added thereto onceevery two days. The cells without the HNF4A gene introduced were used asa control group.

The combination of the genes introduced is shown in Table 1.

TABLE 1 Combination of introduced genes Gene Introduction Time 1 2 3 4 56 7 8 day 3 — SOX17 — — SOX17 SOX17 — SOX17 Mesendodermal cell day 6 — —HEX — HEX — HEX HEX Endodermal cell day 9 — — — HNF4A — HNF4A HNF4AHNF4A Hepatic stem cell

Test Example 12 Results Obtained by Real-Time PCR Method

On the 18th day of culture, the expression amounts of possiblehepatocyte markers ALB, GYP2D6, CYP3A4 and CYP7A1 were determined by areal-time PCR method by using TaqMan® Gene Expression Assays (AppliedBiosystems, catalog number: Hs00910225_m for albumin (ALB),Hs02576168_g1 for GYP2D6, Hs00430021_m1 for CYP3A4 and Hs00167982_m1 forCYP7A1). The expression amounts were calculated, based on the standard(100) of the expression amounts of respective genes in human adult livertotal RNA (Clontech, catalog number 636531). The results are shown inFIG. 30. The number on the abscissa in FIG. 30 represents thecombination of genes shown in Table 1. As a result, albumin, CYP2D6,CYP3A4, CYP7A1 were expressed in drastically increased amounts in thecells containing all three SOX17, HEX and HNF4A genes introduced byusing an Ad vector, compared to the case when other combinations ofgenes were introduced, and thus, it was possible to obtain hepatocytesthat give the drug-metabolizing enzyme in an amount greater than normalcells.

INDUSTRIAL APPLICABILITY

As described above in detail, it was shown that it is possible by usingthe Ad vector according to the present invention to introduce genesassociated with induction of differentiation of pluripotent stem cellssuch as ES or iPS cells effectively and to induce differentiation tohepatocytes. Specifically, it is possible to induce differentiation tohepatocytes effectively by introducing one or more genes selected fromHEX, HNF4A, HNF6 and SOX17 genes into stem cells such as ES or iPScells. It was found that it is possible to induce differentiation fromstem cells to hepatocytes effectively, especially by introducing a HEXgene into stem cells. It was also found that it is possible to induce ofdifferentiation from stem cells to hepatocytes more effectively byintroducing for example SOX17 gene and/or HNF4A gene additionally, asneeded, according to progress of cell differentiation.

It is possible for example to perform evaluation of pharmaceuticaltoxicity and pharmacokinetics by using the hepatocytes prepared by themethod of differentiation according to the present invention.Specifically, it is possible to predict toxicity of a candidate drugcompound in the body previously by adding the candidate drug compound tothe hepatocytes prepared according to the present invention andanalyzing the fluctuation in expression of hepatotoxicity markers. It isalso possible to predict the pharmacokinetics (metabolites) of acandidate drug compound previously by adding the candidate drug compoundto the hepatocytes prepared according to the present invention andanalyzing the metabolites of the compound. It is possible in this way toscreen rapidly candidate drug compounds that are to be eliminatedbecause of their problems in toxicity and pharmacokinetics, thusaccelerating drug development.

If it is possible to induce differentiation from pluripotent stem cellssuch as ES or iPS cells to hepatocytes by the method according to thepresent invention, it becomes possible to reconstruct the hepatocytestructure in vitro and further it may be possible to prepare maturehepatocytes and further the liver itself by differentiation induction.It is possible by using the Ad vector according to the present inventionto induce differentiation from various ES or iPS cells to hepatocyteswithout preparation of a stabilized strain expressing a particular gene.It is thus possible to prepare the individual liver specific to thepatient rapidly if iPS cells are available. In addition, the methodaccording to the present invention may pave a way to treatment byregenerative medicine of diseases that can be treated only with organtransplantation traditionally and thus, it is quite useful.

1-14. (canceled)
 15. A method of differentiating a stem cell into ahepatocyte, comprising: initiating differentiation of the stem cell byculturing the stem cell in a differentiation medium to form an initiatedcell; transforming the initiated cell with a first adenoviral vectorcomprising a first hepatocyte differentiation gene to produce a singlytransformed cell; transforming the singly transformed cell with a secondadenoviral vector comprising a second hepatocyte differentiation gene toproduce a doubly transformed cell; and culturing the doubly transformedcell to form a hepatocyte, wherein the first and second hepatocytedifferentiation genes comprise two or more of HEX, HNF4A, HNF6, andSOX17 genes, and the first and second hepatocyte differentiation genesare not the same.
 16. The method of claim 15, wherein the firsthepatocyte differentiation gene is SOX17 or HEX, the second hepatocytedifferentiation gene is HEX or HNF4A.
 17. The method of claim 15,wherein the first hepatocyte differentiation gene is SOX17 and thesecond hepatocyte differentiation gene is HEX.
 18. The method of claim15, wherein the initiated cell is transformed with the first adenoviralvector at a mesendodermal cell stage.
 19. The method of claim 15,wherein the singly transformed cell is transformed with the secondadenoviral vector at an endodermal cell stage.
 20. The method of claim15, wherein the doubly transformed cell expresses fetoprotein (AFP),albumin (ALB), or both, at a level higher than that of the initiatedcell or the stem cell.
 21. The method of claim 15, wherein the doublytransformed cell expresses α-fetoprotein (AFP), albumin (ALB), CYP2D6,CYP3A4, CYP7A1, or any combination thereof at a level higher than thatof the initiated cell or the stem cell.
 22. The method of claim 15,wherein the stem cell is a human stem cell.
 23. The method of claim 15,wherein the stem cell is an embryonic stem cell or an inducedpluripotent stem cell.
 24. The method of claim 15, wherein the stem cellis a human embryonic stem cell or a human induced pluripotent stem cell.25. The method of claim 15, further comprising contacting at least oneof the stem cell, the initiated cell, the singly transformed cell, andthe doubly transformed cell with at least one humoral factor.
 26. Themethod of claim 25, wherein the at least one humoral factor comprisesactivin A, bFGF, BMP4, FGF-4, HGF, dexamethasone, oncostatin M, or acombination thereof.
 27. The method of claim 15, wherein at least one ofthe initiated cell and the singly transformed cell is cultured for atleast three days before transformation with the first or secondadenoviral vector.
 28. The method of claim 15, further comprisingtransforming the doubly transformed cell with a third adenoviral vectorcomprising a third hepatocyte differentiation gene to produce a triplytransformed cell, and wherein the culturing further comprises culturingthe triply transformed cell to form the hepatocyte.
 29. The method ofclaim 28, wherein the first hepatocyte differentiation gene is SOX17,the second hepatocyte differentiation gene is HEX, and the thirdhepatocyte differentiation gene is HNF4A.
 30. The method of claim 28,wherein the doubly transformed cell is transformed with the thirdadenoviral vector at a hepatic stem cell stage.
 31. The method of claim28, wherein the doubly transformed cell expresses α-fetoprotein (AFP),albumin (ALB), CYP2D6, CYP3A4, CYP7A1, or any combination thereof at alevel higher than that of the initiated cell or the stem cell.
 32. Themethod of claim 28, wherein the doubly transformed cell is cultured forat least three days before transformation with the third adenoviralvector.
 33. A method of differentiating a stem cell into a hepatocyte,comprising: initiating differentiation of the stem cell by culturing thestem cell in a differentiation medium including at least one humoralfactor to form an initiated cell; transforming, at a mesendodermal cellstage, the initiated cell with a first adenoviral vector comprising aSOX17 gene to produce a singly transformed cell; transforming, at anendodermal cell stage, the singly transformed cell with a secondadenoviral vector comprising a HEX gene to produce a doubly transformedcell; and culturing the doubly transformed cell to form a hepatocyte,wherein the doubly transformed cell expresses α-fetoprotein (AFP),albumin (ALB), CYP2D6, CYP3A4, CYP7A1, or any combination thereof at alevel higher than that of the initiated cell or the stem cell, andwherein the stem cell comprises a human embryonic stem cell or a humaninduced pluripotent stem cell.